JP7309728B2 - Acousto-optic device and method - Google Patents

Description

The present invention relates to the field of optical microscopy, in particular multiphoton microscopy.

The present invention relates to an apparatus for reducing the color spread angle of diffracted light in an acousto-optic element, comprising an acousto-optic element and two focusing optics. Furthermore, the present invention relates to a method for reducing the color spread angle of diffracted light in an acousto-optic device.

One key challenge in many areas of optical microscopy is to provide excitation light having one or more preset wavelengths, regardless of the method used. Depending on the type of microscopy and/or depending on the type of sample, one or more excitation light beams, which should generally have preset spectral characteristics, may be required.

For example, in the field of fluorescence microscopy, it is important to use light with wavelengths that excite fluorescence. Different wavelengths are used, in particular if the sample contains fluorescent substances with different excitation spectra.

Of particular importance in the fields of multiphoton microscopy and confocal scanning microscopy (confocal microscopy) is to adapt the intensity for particular wavelengths or to turn particular wavelengths on or off.

For this, wavelength selective elements based on acousto-optic effects can be used. Such an acousto-optic device generally comprises a so-called acousto-optic crystal, which is vibrated by an acoustic signal generator, also called a transducer or "transducer". Generally, such transducers have a piezoelectric material and two or more electrodes contacting the material. By electrically connecting these electrodes to a high frequency, typically in the range of 10 MHz to 10 GHz, the piezoelectric material can be excited to vibrate, thereby generating acoustic waves that pass through the crystal. be. Acousto-optic crystals are distinguished by the fact that the optical properties of the crystal change due to this generated acoustic wave. In particular, a periodic modulation of the local refractive index takes place. This modulation can act like a (Bragg) grating and diffract light of the corresponding wavelength.

Examples of such acousto-optical devices are acousto-optical modulators (AOMs), acousto-optical deflectors (AODs), acousto-optical tunable filters (AOTFs). , an acousto-optical beam splitter (AOBS) and an acousto-optical beam mixer (AOBM). Within the framework of this specification, by AOM generally is meant an acousto-optic element that changes or modulates the frequency and/or the direction of propagation and/or the intensity of incident light depending on the acoustic waves formed in the acousto-optic element. It shall be. For example, AOD represents a special form of AOM dedicated to changing the direction of propagation, while AOTF is an AOM that specifically exploits the filter properties of acousto-optic modulators. The relatively fast modulatable nature of acoustic waves and the low cost compared to other techniques (e.g., by electro-optical action) have made AOMs a standard tool for light modulation in laser scanning microscopy. ing.

AOM is widely known in multiphoton microscopy. Femtosecond lasers with wavelengths in the red to near-infrared region are generally used here (F. Helmchen and W. Denk, "Deep tissue two-photon microscopy", Nature Methods 2, 932-940 ( 2005)). Typical values are wavelengths of 650 nm to 1300 nm, pulse widths of 50 fs to 200 fs, and pulse energies of a few 100 nJ at repetition rates on the order of 100 MHz and linear polarization.

Depending on the field of use and requirements, the AOM and the acoustooptic device or system in general can be optimized based on various parameters. The design parameters are, in particular,
・Color collinearity of plus/minus first order diffracted light (so-called usable beam) ・Diffraction efficiency ・Spectral bandwidth (spectral width) of light efficiently diffracted at the applied high frequency
• Angular sensitivity of the input beam. • High frequency frequency range and amplitude range.

In particular, the chromatic collinearity of the plus/minus 1st order diffracted light (hereinafter also referred to as ±1st order diffracted light for short), ie the collinearity of the utilized beams of different wavelengths, is more important for use in multiphoton microscopy. In other words, within the framework of this specification, the chromatic collinearity of the ±first order diffracted light is the positive first order diffracted light but with different wavelengths, or the negative first order diffracted light but with different wavelengths. means the collinearity of This definition of collinearity also applies to higher orders of diffracted light (ie generally ±nth diffracted lights, where n≧1).

When the AOM is hit by a light pulse with a given center wavelength λ and spectral width Δλ, ±first order diffracted light is formed which is commonly used as a useful beam. Due to the finite diffraction efficiency, it is usually also possible to detect the 0th diffracted light, but the 0th diffracted light cannot be modulated well by the AOM compared to the ±1st diffracted lights, so it is not commonly used. .

Due to the finite spectral bandwidth of the light pulse, the acoustooptic device provides an angular resolution of the ±first order diffracted light. That is, the different color components of the light pulse are diffracted in slightly different directions by the acousto-optic element. Therefore, there is no chromatic collinearity of the ±1st order diffracted lights. What has been said in the paragraphs above, of course, applies equally to higher orders of diffracted light.

The magnitude of this spread by the acousto-optic element is hereinafter referred to as a chromatic spread angle (CSA), and defined as the differential of the diffraction angle φ of the diffracted light with respect to the wavelength λ of the diffracted light. That is, CSA=dφ/dλ. That is, CSA is a measure of the strength of deviation from perfect chromatic collinearity within one diffraction order and has units of mrad/nm. The wider the spectral bandwidth of the light incident on the acousto-optic device, the wider the angular range over which the light is diffracted (for a given CSA). Therefore, it is particularly desirable to compensate or reduce CSA in light with a wide spectral bandwidth in order to efficiently utilize the diffracted light. This is because with the CSA this angular range also narrows.

Too large a CSA for use in (multiphoton) microscopy is problematic for two reasons. First, the different directions of propagation of the color components of the femtosecond light pulses lead to elliptical beam profiles as they propagate, and thus suboptimal illumination of the entrance pupil of the excitation objective, which In particular, either the resolution will be compromised or the output will be degraded (if the entrance pupil is over-illuminated). Second, different color components of the light pulse hit the entrance pupil at different angles, which results in these color components being spatially separated in the sample. This spatial separation (also called "spatial chirped light pulse" or "spatial chirp") reduces the peak intensity as well as the non-simultaneous arrival of the color components ("temporal chirp"), thus reducing multiphoton excitation. Efficiency is also reduced.

The drawback caused by the first point, which produces a propagation interval dependent flattening (or "spatial chirp"), can be compensated by the prior art, which is the virtual reciprocal of the AOM by suitable relay optics. This is done by imaging the point of action onto the entrance pupil of the objective lens and by causing no oblateness. Within the framework of this specification, a virtual interaction point (diffraction point) of an acousto-optical element, in particular of an AOM, is understood to mean the beam course of a plurality of beams emitted by the acousto-optical element with the physical direction of propagation of the light. is understood to be the single point obtained when extending inversely and identifying their points of intersection.

In particular, to solve the second problem ("temporal chirp"), the prior art uses a prism (S. Zeng et al., "Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism ", Opt. Lett. 31, 1091-1093 (2006)), a grating or another acousto-optic component (Y. Kremer et al., "A spatio-temporally compensated acousto-optic scanner for two-photon microscopy providing large field of view. ", Opt. Express 16, 10066-10076 (2008)) have been used, which in a suitable arrangement substantially allow the CSA caused by the AOM to be compensated back. However, these approaches are either relatively costly or lack flexibility (eg, if the laser wavelength changes).

It is therefore an object of the present invention to significantly reduce the above-mentioned CSA of light diffracted by acousto-optic devices, in particular AOMs, thereby enabling high-quality multiphoton microscopy in a cost-effective manner. The goal is to show a device that is versatile and flexible. A further object of the invention is to specify a corresponding method.

Unlike the above solution approaches of the prior art, which compensate for the spectral content of the light beam or laser pulse in position and orientation with respect to the acousto-optic element by suitable optical components, the present invention provides Only color component collinearity is guaranteed. These color components are then imaged at the same position on the sample side.

Based on this approach according to the invention, we have found the technical implementation of claim 1 for a lens system that is surprisingly simply constructed and thus cost-effectively manufacturable. The lens system can be flexibly operated in conjunction with acousto-optic elements and meets high quality requirements for CSA compensation of diffracted light. Advantageous embodiments are the subject matter of the dependent claims as well as the following description.

According to a first embodiment of the invention, the inventive device for reducing the color spread angle of diffracted light in an acousto-optic element comprises an acousto-optic element arranged in the optical path of an incident light beam, the acoustic The optical element forms from the incident light beam diffracted light originating from a virtual interaction point of the acousto-optic element, the device further comprising two focusing optics, a first focusing The optical system is arranged in the optical path in front of the acousto-optic element, the second focusing optical system is arranged in the diffracted light, and the focal point of the incident light beam behind the first focusing optical system is the acoustic Located on the optical element, the virtual interaction point is characterized in that it is at the front focus of the second focusing optics.

In principle, it is preferable to implement the invention with an optical system having the shortest possible focal length. as this reduces color eccentricity. However, this can lead to a more or less significant reduction in diffraction efficiency, depending on the design of the acousto-optic element each time. That is, the larger the particular angle of incidence of the incident light (the angle between the optical axis and the beam path), the more inefficiently the incident light is generally diffracted by the acousto-optic element. In practice, therefore, a compromise must be made between eccentricity and diffraction efficiency in consideration of the specific application.

By means of a second optical system with a focal length distance to the virtual interaction point and behind the acousto-optic element, the color components (here color component means diffracted light with a particular wavelength) are , propagate substantially collinear to each other and collinear to the optical axis. However, such an arrangement focuses the color components or the resulting light beams, thus indirectly leading to divergence of the resulting light beams, thus counteracting this divergence of the light beams. A first optical system in front of the is required. This first optical system in front of the acousto-optic element focuses the incident light beam so that each wavelength of the diffracted light (i.e. all color components) is distributed to the smallest possible beam diameter at the acousto-optic element. to have

The device according to the invention can be used particularly advantageously with pulsed incident light beams having particularly short pulse durations. This is because the shorter the pulse duration, the wider the spectral width and thus the greater the angular range over which the light is diffracted. Thus, reducing CSA is necessary or at least desirable. The application of this device in multiphoton microscopy or in the field of multiphoton microscopy is mentioned here by way of example, where the pulse duration of the light utilized is typically in the range of a few hundred femtoseconds. is used. Therefore, according to another embodiment of the present invention, the incident light beam has a maximum A laser light source emitting pulsed light (pulsed laser light source, pulsed laser light source, pulse laser, pulsed laser).

According to a third embodiment of the invention, the diffracted light is plus/minus first order diffracted light. This is because this order of light is preferably used for microscopy applications.

The above principle can be implemented, for example, by cylindrical lenses and/or by spherical lenses. An advantage when using cylindrical lenses is that the peak intensity (maximum intensity) at the crystal, and especially at the crystal surface, is relatively small. Achromatic and/or aspherical lenses, for example, are also conceivable to improve the imaging properties. It is also possible to implement the described invention with curved mirrors, which has the advantage over the use of standard lenses that no chromatic aberration occurs. Such mirrors can be designed with a spherical structural form or (to avoid spherical aberration) as parabolic mirrors. For geometrical reasons, parabolic mirrors in the form of "off-axis" paraboloid (OAP) are also suitable in some situations. In general, the optical system used may include one or more lenses and/or mirrors and/or other optical elements such as filters or prisms, a very wide variety of combinations being possible.

Therefore, according to another preferred realization of the invention, the first focusing optics and/or the second focusing optics comprise at least one mirror and/or at least one lens.

According to a realization of the device according to the invention, which is particularly favorable because of its structural simplicity and cost advantages, the first focusing optics and/or the second focusing optics are lenses, in particular spherical lenses. Alternatively, it is composed of a cylindrical lens. Using a cylindrical lens is advantageous because this AOM breaks the rotational symmetry of the beam course. This is because the AOM causes decomposition in only one spatial direction. If the two focusing optics are realized by individual lenses, these optics preferably either both consist of spherical lenses or both consist of cylindrical lenses. This ensures that the beam is not unnecessarily focused or diverged in one dimension.

As already mentioned above, the invention is particularly preferably implemented using an acousto-optic modulator (AOM).

As explained above, the device according to the invention is particularly suitable for use in microscopy and in microscopy in the field of scanning microscopy, in particular multiphoton microscopy. Therefore, according to another embodiment of the invention, a microscope, in particular a multiphoton microscope, comprises a device according to any one of claims 1-7.

A microscope (or microscope system) equipped with such a device (e.g. a multiphoton microscope) includes a pulsed light emitting laser source for illuminating the sample, which laser source has a maximum output of 1000 fs ( ie ≤ 1000 fs), preferably up to 800 fs (≤ 800 fs), preferably up to 500 fs (≤ 500 fs), preferably up to 300 fs (≤ 300 fs), particularly preferably up to 200 fs (≤ 200 fs), also very preferably can form a light beam incident on the device with a pulse duration of up to 100 fs (≦100 fs). The device according to the invention reduces the CSA of the diffracted light at the acousto-optic element of the device, which is formed from the incident pulsed light beam. In this connection, it should be noted again that as the pulse duration becomes shorter, the spectral width and thus the angular range over which the light is diffracted becomes wider, i.e. for short pulse durations, the device according to the invention is particularly advantageous for reducing CSA.

The method according to the invention for reducing the Chromatic Spread Angle (CSA) of diffracted light diffracted by an acousto-optic element preferably comprises a device according to any one of claims 1 to 7. is carried out using

With knowledge of the above-described device features of the solution according to the invention, a person skilled in the art working in the field of the invention can adapt the device features in order to preferably use the above-described device. would constitute method steps. In this regard, to avoid repetition, reference is made to the preceding portions of the specification.

There are various options for advantageously configuring and developing the teachings of the present invention. In this regard, reference is made to the claims subordinate to claim 1 on the one hand and to the following description of the invention based on the drawing on the other hand. Generally preferred embodiments and developments of this teaching are also described in connection with the description of preferred embodiments of the invention based on the drawings.

1 is a schematic diagram showing a typical illumination light path of a scanning microscope; FIG. 1 is a schematic diagram showing diffraction processes in an AOM; FIG. 1 is a schematic diagram of an apparatus according to the invention; FIG. FIG. 5 is a partial schematic diagram showing the beam course when using a cylindrical lens as the second optical system;

FIG. 1 shows in greatly simplified form the typical illumination beam path of a scanning microscope, for example a multiphoton microscope, with an acousto-optical element 1, in particular an AOM, as beam modulation means. The incident light beam 2 originates from a light source 22 , for example a laser, in particular a pulsed laser, and reaches the acoustooptic device 1 optionally via beam guide optics and adaptation optics 24 . From here, the diffracted light 7 in the acoustooptic element 1 reaches the objective lens 28 via selective imaging and scanning optics 26 and finally reaches the sample 30 .

FIG. 2 schematically shows the diffraction process in an AOM 1 impinged by an incident light beam 2 with a given center frequency λ and spectral width Δλ. Attached to the crystal of AOM 1 is a transducer 5 that applies radio frequency (RF) to the crystal. In the resulting grating, the ±first orders of the diffracted light 7 are formed, which are generally used as a useful beam. Due to the finite diffraction efficiency, the 0th order diffracted light 8 is also generally detectable. The imperfect chromatic collinearity of the first order diffracted light and the finite spectral bandwidth of the incident light pulse 2 give rise to an angular resolution 9 of the ±first order diffracted light, in which the different color components of the light pulse are separated into only a few are diffracted in different directions. This is shown by way of example for three partial beams 7a, 7b, 7c with different wavelengths λ ₁ , λ ₂ and λ ₃ . The magnitude of the spread of the diffracted light is expressed in units of mrad/nm by the chromatic spread angle (CSA). These diffracted partial beams 7a, 7b, 7c originate from a virtual interaction point 50 of AOM1, which point of interaction 50 corresponds to any refraction at the transition between the crystal of AOM1 and the surrounding medium. Without taking into account the effect, it is obtained if the beam course is extended against the physical direction of propagation and the point of intersection is specified.

FIG. 3 shows a schematic diagram of an apparatus according to the invention, in which a first optical system 20 (for example a separate lens) in front of AOM 1 and a second optical system 10 ( are also provided, for example separate lenses). The first optical system 20 is arranged such that the light beam of the incident light 2 focused by this first optical system 20 is focused at the AOM 1 to the focal point 40 at a distance d from the first optical system 20, i.e. The beam paths of the light beams of the incident light 2 are positioned such that they intersect at one point in the crystal. (In this figure, the refraction effects as light travels from the surrounding medium to the crystal are not depicted for clarity. However, this should of course be taken into account when arranging the first optical system 20. The distance d generally does not correspond to the focal length of the optical system 20 in the surrounding medium, e.g. Also, for example, the color components of the ±1st-order diffracted lights are positioned so as to progress collinearly with each other (collinearity). These color partial beams 7a, 7b, 7c transition into partial beams 17a, 17b, 17c. The collimated beams (including the partial beams 17 a , 17 b , 17 c ) generated in this way are directed to the sample 30 . The distance between the second optical system 10 and the virtual interaction point 50 thus corresponds to the focal length f of the second optical system 10 . Here the second optical system 10 is used to obtain the chromatic collinearity of the ±first order diffracted light and thus the collimation of the diffracted light, whereas the first optical system 20 is used for the chromatic part Undesired divergence of the beams 17a, 17b, 17c (see FIG. 4) and of the resulting light beams is compensated as far as possible, or at least used to counteract this .

4 shows schematically and partially in detail the beam course when using a cylindrical lens as the second optical system 10, in the partial views a) and b) 90° to each other with respect to the optical axis 3. The arrangement is shown from two perspectives rotated by degrees. The color partial beams 7a, 7b, 7c are each focused by a cylindrical lens 10, which in the spatial direction behind the cylindrical lens 10 results in an undesired divergence of the partial beams 17a, 17b, 17c and the consequent An undesired divergence of the light beam occurs (see partial view a)). This divergence is compensated as much as possible by the first optical system 20 in front of the AOM 1, which compensation is due to the focusing effected by this first optical system 20 in FIG. This is done by ensuring that the diffracted light (ie color components) of each wavelength exemplarily delineated by the partial beams 7a, 7b, 7c each itself has the smallest possible beam diameter.

Claims (19)

A microscope comprising a device for reducing the color spread angle of diffracted light (7) in an acousto-optical element (1),
The device comprises the acousto-optic element (1) arranged in the optical path of an incident light beam (2),
The acousto-optic element (1) forms diffracted light (7) originating from a virtual interaction point (50) of the acousto-optic element (1) from the incident light beam (2),
said microscope comprising a laser light source emitting pulsed light for illuminating a sample, said laser light source providing said incident light beam (2) having a pulse duration of ≦1000 fs;
The device includes two focusing optics (10, 20),
A first focusing optical system (20) is arranged in the optical path in front of the acoustooptic element (1), and a second focusing optical system (10) is arranged in the diffracted light (7). cage,
the focal point (40) of the incident light beam (2) behind the first focusing optics (20) is located at the acousto-optic element (1);
said virtual interaction point (50) is at the front focus of said second focusing optics (10);
microscope . The diffracted light (7) is plus/minus first order diffracted light,
A microscope according to claim 1. said first focusing optic (20) and/or said second focusing optic (10) comprises at least one mirror;
3. A microscope according to claim 1 or 2 . said first focusing optic (20) and/or said second focusing optic (10) comprises at least one lens;
4. A microscope as claimed in any one of claims 1 to 3 . said first focusing optic (20) and/or said second focusing optic (10) consists of one lens,
5. A microscope as claimed in any one of claims 1 to 4 . Said first focusing optics (20) and/or said second focusing optics (10) consist of one spherical or cylindrical lens,
6. A microscope according to claim 5. The acousto-optic device is an acousto-optic modulator,
7. A microscope as claimed in any one of claims 1 to 6. wherein the microscope is a multiphoton microscope;
8. A microscope as claimed in any one of claims 1 to 7 . said laser light source providing said incident light beam (2) having a pulse duration of ≦ 300 fs;
9. A microscope as claimed in any one of claims 1 to 8 . said laser light source providing said incident light beam (2) having a pulse duration of ≦100 fs;
9. A microscope as claimed in any one of claims 1 to 8. A method for reducing the color spread angle of diffracted light (7) in an acoustooptic device (1) using a microscope according to any one of claims 1 to 10 , comprising:
The acoustooptic element (1) is placed in the optical path of an incident light beam (2), the diffracted light (7) is formed from the incident light beam (2), the diffracted light (7) is starting from a virtual interaction point (50) of said acoustooptic element (1),
The method includes
focusing the incident light beam (2) to a focal point (40) located at the acousto-optic element (1) by a first focusing optics (20);
forming said diffracted light (7) from said incident light beam (2) by said acousto-optical element (1), said diffracted light (7) being a virtual mutual starting from the point of action (50);
passing said diffracted light (7) through a second focusing optic (10), wherein said virtual interaction point (50) is at the front focus of said second focusing optic (10); a step located
including
said incident light beam (2) is a pulsed light beam from a laser light source emitting pulsed light with a pulse duration of ≦1000 fs;
Method. said incident light beam (2) is a pulsed light beam from a laser light source emitting pulsed light with a pulse duration of ≦ 300 fs;
12. The method of claim 11 . said incident light beam (2) is a pulsed light beam from a laser light source emitting pulsed light with a pulse duration of ≦100 fs;
12. The method of claim 11. The diffracted light (7) is plus/minus first order diffracted light,
A method according to any one of claims 11-13 . said first focusing optic (20) and/or said second focusing optic (10) comprises at least one mirror;
A method according to any one of claims 11 to 14 . said first focusing optic (20) and/or said second focusing optic (10) comprises at least one lens;
A method according to any one of claims 11 to 15 . said first focusing optic (20) and/or said second focusing optic (10) consists of one lens,
A method according to any one of claims 11-16 . The first focusing optics (20) and/or the second focusing optics (10) consist of one cylindrical lens,
18. The method of claim 17. The acousto-optic device is an acousto-optic modulator,
A method according to any one of claims 11-18 .

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